Structures and Mechanisms - ACS Publications - American Chemical

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Preface A visitor to Professor William N . Lipscomb is exposed to both a great legacy of accomplishments and to proposals for new multiyear scientific explorations that are outlined with the excitement of a be ginning assistant professor. This pairing is a large part of the explanation for why a group of prominent scientists gathered at Harvard on May 1214, 2000, to somewhat belatedly celebrate the 80 birthday of "the Colonel." This book is in no way a "proceedings" of that scientific cele bration, but the contributors were invited from among those who have studied with the Colonel as undergraduates, graduate students, postdoc toral research associates, and visiting faculty. The chapters in this book purposely reflect the fact that Lipscomb and his co-workers have con tributed to a wide range of chemistry. We believe that this is one of the great strengths of the Lipscomb tradition, and it is intended that the breadth of the book be stimulating, and cause readers to think a little beyond the usual confines of a particular research field. We hope that the juxtaposition of, for example, boron hydrides, quantum mechanical calculations, and structure of a virus will provide a perspective on chem istry not offered by traditional texts. th

An autobiographical reflection by Lipscomb, which he wrote in 1977, is included with the front matter of the book. The book begins with an introduction to Lipscomb's science by one of the editors. Following this introductory section, there are three sections of scientific reviews. The first section focuses on the "inorganic" chemistry legacy, beginning with Russell Grimes' essay on polyhedral boranes, and continuing with increasing quantum-mechanical emphasis. Because patterns have been a central theme of the Lipscomb contributions in inorganic chemistry, we end this section with Irving Epstein's chapter on oscillations, waves, and patterns in chemistry and biology. The Colonel repeatedly emphasized that theory could lead experiment, and the next section presents chemical theory of bonding and N M R parameters, and includes, appropriate to the scope of the Lipscomb legacy, interference of atom lasers by Roger Hegstrom. The final section shows the legacy of the third phase of Lips comb's scientific career, the relation of protein structure to function. The

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first chapter in this section is by Martha Ludwig, who participated in the early work on the structure of carboxypeptidase. Douglas Rees discusses biological energy transduction, Florante Quiocho discusses proteinligand interactions, and Tomas Steitz relates structures to the central dog ma of biology. In addition to crystallography, structure determination by N M R and EPR is included. This section ends with comments on the relation of the retrovirus core to the function of this molecular machine by Lipscomb's last Ph.D. student, Karen Reinisch. Thanks to the authors for the pleasure that they have given us via their chapters. If this book serves as even a mild antidote to the seemingly relentless pressure for a narrower focus in modern science, that will justify to the editors that putting this book together was not merely intellectual hedonism. The calligraphy in the introduction was created by Jean Evans (wife of the Colonel) for this book. We thank her for this contribution. Just as this book was nearing completion, our colleague Don Wiley tragically disappeared while attending a scientific advisory board meeting of St. Jude's Children's Research Hospital in Memphis, Tenn essee. At that time, we had not yet mutually agreed upon a title. Don's latest communication to us on this topic was: "I like the title that ends Ashes to Enzymes. It is so biblical." Consequently, we decided to use that title.

Gareth R. Eaton Department of Chemistry and Biochemistry University of Denver Denver, C O 80208-2436

Don C. Wiley Department of Molecular and Cellular Biology Harvard University 7 Divinity Avenue Cambridge, MA 02138

Oleg Jardetzky Stanford University School of Medicine 300 Pasteur Drive R-320 Stanford, C A 94305-5337

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Process of Discovery (1977); An Autobiographical Sketch


William N. Lipscomb

History

Early experiences My early home environment, on the outskirts of Lexington, Kentucky from 1922 to about 1940 stressed personal responsibility and self reliance. Independence was encouraged especially in the early years when my mother taught music and when my father's medical practice occupied most of his time. In those days general practitioners still existed, and physicians even made house calls. Science hardly existed in the Victorian-like Sayre elementary school, but I studied somewhat independently, learning the distances between objects in the solar system having already become fascinated with the night sky. Also, I collected animals, insects, pets, rocks and minerals in those grade school days. Part of me will always remain close to the country. My interest in astronomy took me to the visitor's nights at the Observatory of the University of Kentucky, where Prof. H. H. Dowing and I became lifelong friends. He gave me a copy of Baker's "Astronomy" which I read many times. I am sure that I gained many intuitive concepts of physics from this book, and from my conversations with him. Within 100 yards, five others of my age group were to emerge two physicists (W. B. Fowler and E. C. Fowler), two physicians (W. R. Adams, J. Adams) and an engineer (R. Fish). Morse-coded messages over a wire stretched high across the street, crystal sets, Tom Swift (the boy Edison-like inventor) books, electric arcs, and lots of talk about astronomy and then chemistry occupied us in a far more interesting way than did the school work. By the age of 12, I had acquired a small Gilbert chemistry set, but rather

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than abandoning it a few weeks after that Christmas day, I set about expanding it partly by ordering apparatus and chemicals from suppliers and partly by using my Father's privilege to purchase chemicals at the local drugstore at a discount. These were the years of the Financial Depression. Of course, I made my own fireworks, and entertained both willing and unwilling visitors with spectacular color changes, vile odors, and explosions with pure hydrogen and oxygen. My tolerant, but concerned mother raised a question only once, when I attempted to isolate a large amount of urea from the natural product. Nevertheless, I had learned a considerable amount of chemistry and physics by the time I enrolled in thefirstchemistry course ever taught at Picadome (now Lafayette) high school. I was a sophomore in a class otherwise restricted to seniors, but ended the year by teaching qualitative analysis to this class. L. Frederick Jones, the teacher, gave me his college books on organic, analytical and general chemistry, and simply asked that I show up for the examinations. I heard most of the classes from the back of the room, where I was doing a bit of laboratory research that I thought was original: the preparation of hydrogen from sodium formate (or sodium oxalate) and sodium hydroxide. This idea was an extrapolation, from the organic text to inorganic chemistry, of the preparation of saturated hydrocarbons, e.g. the preparation of methane from sodium acetate and sodium hydroxide. This study was carefully and thoroughly done, including gas analyses and searches for probable side reactions. I later had a physics course, and took first prize in the state contest on that subject. The library was (understandably) poor in those days in a county high school, but I remember being influenced so strongly by Abbott's "Flatland" that I became very interested in special relativity. Also, I repeatedly read Alexis Carrel's "Man the Unknown," and perused unsystematically the large medical texts in my father's library. While I had no desire to become the fourth generation of physicians in the family, this influence and that of Linus Pauling years later led to my biochemical studies of recent years. I also pursued my independent study program during my busy years (193741) including a music scholarship at the University of Kentucky. Dushman' s "Elements of Quantum Mechanics," the Pittsburgh Staff book on "Atomic Physics" and Pauling's "Nature of the Chemical Bond" were of direct interest. Perhaps I learned more from attempting to help my fellow students than from any other source. Mathematics was a strong interest, considerably heightened by Prof. Fritz John under whom I was the only student in a summer course in Vector Analysis. We spent much of the time pursuing Maxwell's equations jointly, and he introduced me to tensors and matrices. In physics, Prof. 0. T. Koppius was an especially warm friend who also taught me thermodynamics, and Prof. Bertrand Ramsey helped me with introductory quantum mechanics. Prof. F. W. Warburton asked me to give the lectures in the summer course in Electricity and Magnetism inasmuch as I was the only student, and he had three other lectures to give every day. Most influential in my early chemistry career

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was Prof. Robert H. Baker. When I finished the most interesting undergraduate course that I experienced, in qualitative organic analysis, he suggested that I take on a research problem: the direct preparation of derivatives of alcohols from dilute aqueous solution without first separating the alcohol and water. I suggested one method, and he suggested another. Both methods worked, leading to my first publication. In physical chemistry, I became interested in conductance of water as a solute in liquid hydrogen fluoride, and especially whether the results indicated the presence of H ^ * * , but I finally abandoned this work as incorrect. Choice of graduate school was upon me in 1940-41. Prof. Maxson, Chairman of Chemistry (U of K) had decided that I would accept the standing fellowship to MIT. Northwestern offered me a research assistantship at $150/month, Caltech offered me a teaching assistantship in Physics at $20/month, and Prof. H. C. Urey then of Columbia University wrote me a nice letter of rejection. I took the Caltech offer, preferably to study theoretical quantum mechanics with Prof. W. V. Houston, but primarily because of the need for a stimulating environment, and for research problems that were more current. After one semester, I switched to the Chemistry Department under the influence of Prof. Linus Pauling to whom I was drawn by his penetrating and imaginative comments at colloquia, almost always made after everyone else had asked their own questions. At this point (December 1941) the war intervened, and for the following four years my efforts were mostly concerned with the National Defense Research Council. The first project was on analysis of smokes according to particle size, related to a project, no longer needed, to obscure the Los Angeles area optimally. However, most of my war work was concerned with nitroglycerin-nitrocellulose propellants, and involved handling pure nitroglycerin on many occasions. Rates of burning, and polarizing microscopic examination were the major areas. During these four years, I also carried out, with Verner Schomaker, several electron diffraction studies on molecular structures of gas molecules, and learned X-ray diffraction methods from Edward W. Hughes, on the C-N distance in methylamine hydrochloride. Hughes suspected that Hendrick's structure was wrong, and Pauling needed a good C-N distance from a simple molecule for his ideas on peptide bonds. I made most of this X-ray study at Caltech while Hughes was in Emeryville. Some of his replies to my research reports begin, "My God, Willie," with good reason. Actually, this is the first paper in which statistical treatment of errors was made in either the least squares or Fourier methods of X-ray crystallography. Pauling's course in the "Nature of the Chemical Bond" was worth attending every year, because each lecture was new, and Tolman's lectures in Thermodynamics were a model of clarity and perception. Finally in January 1946, I resumed a normal program, and finished my Ph.D. that summer. Especially noteworthy were courses from Prof. J. H. Sturdivant on X-ray

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diffraction and one on particle physics by Prof. J. R. Oppenheimer. My research orientations of later years were strongly influenced by the structural chemistry approach, by Pauling's interest in antigen-antibody (hapten inhibition) studies, by his lectures on chemical bonding especially in boron hydrides and metals, and by the unique certainty of X-ray diffraction results coupled with the less secure interpretation of chemical behavior,froma structural point of view. I tried always to imagine what was happening to structures or reactions, in three dimensional terms. While I was able to use both the mathematical description and the more symbolic chemical symbolism, I returned continually to the structural interpretations whenever I could do so.

Minnesota. 1946-1959 By early 1946 I had applied for a National Research Council Fellowship to study crystallography at low temperatures with Bert Warren of MIT. I had perceived this area as nearly untouched, and full of promising problems such as the boron hydrides, hydrogen bonding, residual entropy studies, and a host of structures for which electron diffraction gave ambiguous or insecure results. However, I withdrew my application in order to accept an assistant professorship at the University of Minnesota at $2,880 per 9 months. My rapture at learning of the rank was modified by the salary. Harold Klug had just left Minnesota for the Mellon Institute, and his apparatus was still there. However, I tossed it all away, and set up a sealed tube unit with the aid of a power supply kindly donated by a local dentist, who demonstrated (on me) how he used hypnosis for his patients. My research started very slowly partly because of the teaching load of two courses, and also because of a lack of research funds. If my memory serves correctly, my support from the Office of Naval Research began in 1948 at a level of $9,000 per year. By then, I had initiated the series of low temperature X-ray diffraction studies first of small hydrogen bonded systems, residual entropy problems and small organic molecules. Later, after the techniques were under control, we studied the boron hydrides themselves B H , B H , B H , B H , B H i , and many more related compounds in later years (50 structures of boron compounds by 1976). 5

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John Gruner, mineralogist at the University of Minnesota, asked me to look into structure of HMn0 , which he had named Groutite in honor of Prof. Frank Grout (U of Minn.). He also dug out some black crystals from a spherical iron pressure-temperature vessel in which some phosphates were also present. Having established that the X-ray powder pattern was similar to that of lazulite, he asked us (Lewis Katz and me) to do a single crystal study. It was certainly a new crystal form, tetragonal instead of monoclinic, and had iron in both II and III valence states. Although artificially prepared, Gruner named it lipscombite. It has since been found a number of locations as a real mineral. 2

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In early 1948 I thought that there was an experimental solution of the phase problem of X-ray crystallography. The idea was to use a double reflection h, followed by h which diffracts in the direction of h = hj + l^. If hj is set on the sphere of reflection so that it diffracts for any orientation of the crystal about a suitably chosen rotation axis, then h, and h should show an interference effect. This idea, beautiful in principle, was defeated by the mosaic character of crystals and possibly also crystal boundary effects. Our experiment in which h is 040 of a glycine crystal failed, although some reflections which were forbidden as single diffractions were observed. Shortly thereafter (1951) Bijvoet published his experimental solution to the phase problem by multiple isomorphous replacement methods, and I thought then that his discovery opened the way to solve protein structures. However, I did not start work in this direction until about 1958, and pursued it seriously beginning in 1961. In the fall of 1953 the structures of B H , B H , B H , and the B , arrangement B H , were known unambiguously from our low temperature single crystal studies. Also, the B H structure was known from thermodynamic infrared studies of Stitt, Longuet-Higgins and Bell, and Price; and the B H structure had been determined by Kasper, Lucht and Harker. All of these boron arrangements were fragments of the two then-known polyhedra, the B octahedron, and the B icosahedron. Our X-ray studies were difficult because of the instability of the preparations and the tendency for explosion if a vacuum line cracked. William Dulmage had such an explosion, which just missed him as he bent over in handling B H on a vacuum line. The folklore regarding H B 0 was not then known: use dry ice for traps, not liquid nitrogen, so that the H B 0 does not accumulate. Maintenance of the liquid nitrogen supply led to emergency trips in cars or streetcars with Dewars to be filled at the local plant, and on occasion led to a watchman's question about whether we were stealing apparatus from the laboratory. The first B H study was terminated when an electrician dropped a wrench on a transformer, shorting it out, and years later (1957) the second B H investigation was ended prematurely when an undergraduate assistant tripped on the stairs in the very early morning, on his way to fill the Dewar, and lay unconscious for about an hour. In puzzling over these structures, I had already noticed the similarity of bonding in B H to that in the bridge regions of B H , B H , B H and B H . These similarities led me to write in the 1954 paper, "These ideas suggest that to a considerable degree the hybridization about boron in many of these higher hydrides is not greatly different from the hybridization in diborane. In addition the probable reason for the predominance of boron triangles is the concentration of bonding electron density more or less towards the center of the triangle so that the bridge orbitals (π orbitals in B H ) of the three boron atoms of the triangle overlap. It does seem very likely from these structural comparisons that the outer orbitals of an atom are not always directed toward the atom to which it is bonded. This property is to be expected for atoms which are just starting to 2

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fill new levels and therefore may be a general property of metals and intermetallic compounds." This paper was a companion to "The Valence Structures of the Boron Hydrides," by W. H. Eberhardt, B. Crawford, Jr. and myself, in which the generality of three center bonds in the boron hydrides was first recognized. We also formulated a rather simple set of rules which gave three relations between the chemical formula B H and the constituent BHB bridges (s), three-center BBB bonds (t), two-center BB bonds (y) and B H groups (x) : s+x = q, s+t = p, and ρ = t+y+q/2. There are three equations and four unknowns (styx) for a given formula so that ambiguities remain even when one requires that s,t,y and χ shall not be negative. After using these equations and structural elements among these and related compounds, I finally decided that in many compounds the ambiguities are quite real, representing different isomers or pathways of transformations. In those days X-ray intensities were measured visually with the use of a standard scale. A major problem was to locate the number and position of hydrogen atoms in these compounds. High quality data were required. For B H even the number of boron atoms was not known until the structure was solved by the method of Holmes (". . . when all other contingencies fail, whatever remains, however improbable, must be the truth." From The Complete Sherlock Holmes, Garden City Publishing Company, Inc. 1089; see also pp. 118, 360, 1192). The number of hydrogen atoms was also established in further X-ray analysis of this material. Another example is the complete formula and structure of B Cl both of which were established by the X-ray data alone. At least our speculations of bonding and reactivity were based on secure structures. "We have even ventured a few predictions, knowing that if we must join the ranks of boron-hydride predictors later proved wrong, we shall be in the best of company" (Eberhardt, Crawford, Lipscomb, 1954). The spirit in which these and related later extrapolations, guesses and predictions were made was the realization that a large body of new chemistry existed, if we could only persuade ourselves and other chemists to look for it. Anyway, it has been fun to guess and to be proved right, as well as distressing to be proved wrong. p

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In 1954-5 on a Guggeheim Fellowship to Oxford, I spent most of my efforts on theoretical chemistry, half-intending to leave both X-ray diffraction and boron chemistry. However, the impressive and highly intuitive, solution of the vitamin B structure was being obtained by Dorothy Hodgkin and her associates there, and I resolved to go to larger structures with the aid of high speed computers when I returned. In boron chemistry, Riley Schaeffer, Herman Schlesinger and, later, M. Frederick Hawthorne sent us new boranes, boron halides and derivatives of boranes for structural studies. As an aid in solving the crystal structures and for predicting new boranes, I developed a very intuitive approach, which Richard Dickerson and I formalized into a topological set of rules. 1 2

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One misses a lot of important discoveries on the way. I remember one day in Oxford in the fall of 1954 when Coulson mentioned the single nuclear magnetic resonance in PF . Donald Hornig, with whom I shared the office, suggested intermolecular exchange in order to produce this unexpected equivalence of the equatorial and axial fluorine atoms, whereupon I suggested an intramolecular rearrangement process. This is, of course, the pseudorotation proposed by Steven Berry a few years later. I did not publish this result, or even think of this area as a new and promising one at that time. In a sense, however, this discussion made it much easier for me to understand the equivalence of all boron and all hydrogen atoms in the B nuclear magnetic resonance spectrum of B H " by use of a pseudorotation process. This occurred in December of 1958 while refereeing a communication by Earl Muetterties and W. D. Phillips, when I found that their incorrect static structure would not give the relative intensities which they had found experimentally. Actually this spectrum was not well above background so that I had to assume that the observed septet was really a nonet in which the two outer lines were weak. Another preparation for this interpretation was the difference in hydrogen positions in B H (four bridges) and B H " then extrapolated from the B i H (NCCH ) structure as a two-bridge, two-BH structure. The detailed mechanism of the B H * rearrangement had to preserve the coupling constant between Β and Η averaged on the nmr time scale. Later I developed a general theory of pseudorotation in polyhedral species (1966). Internal rearrangement in B H was foreshadowed by the ambiguity in the hydrogen arrangement (J. Inorg. Nucl Chem. 11, 1 (1959)), in an article in which I attempted to systematize the chemical transformations then known for the boranes. One rather successful prediction was the probable existence of B H " and B H , which have framework orbitals like those in B H and indeed have a σ, π bonding scheme rather like that in the aromatics C H ", C H and C H . Both B H and B H were eventually prepared in other laboratories. Many times, following the 1954 ECL paper, I decided to leave boron chemistry, only to find myself thinking of a new way to look at the structures, or hearing of a new interesting preparative success. It was and still is, an area of chemistry to which I returned almost subconsciously within half a year of each decision to move on to other research areas. The most important other direction was the determination of large organic structures, preferably natural products. In 1953 I wrote, "The elucidation of complete molecular structures of relatively complex organic compounds, often by methods which require a minimum of chemical information, by X-ray diffraction techniques has been one of the most significant advances in recent years. The total effort in man hours required for such a complete structure determination is sometimes less that expended in the determination of the molecular structures by the more classical organic methods. Lack of realization of this fact by most organic chemists is matched only by the reluctance of most crystallographers to under-take such 5

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investigations" (Ann. Rev. Phys. Chem. 1953). This optimism was only later to be fully realized with the advent of the high speed digital computer and the automated X-ray diffractometer. Our studies of natural products in my later years at Minnesota were a prelude to later work at Harvard in the three dimensional structure and function of enzymes. Of the many other studies done at Minnesota, I may mention the study of Roussin's black salt (Fe S (NO) ",Cs ) from which I failed to realize that the Fe S cluster later found in oxidation-reduction proteins, might be a reasonable extrapolation. On the more humorous side, I comment on the SiF structure, obtained when I tried to do the HF structure in pyrex capillaries (later we did solve the HF crystal structure), and the cyclic silane (Si0 ) obtained from a vacuum line preparation of boron hydrides in which silicone stopcock grease was used. Also, countless single crystals of ice were photographed from condensate on the outside of our capillaries, which were cooled to low temperatures for X-ray diffraction work. However, we took full advantage of the low humidity of the Minnesota winter by opening the windows of the X-ray laboratory. +

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Harvard 1959 While not at all unhappy at Minnesota, I was aware that the Harvard Chemistry Department was one of excellence and certainly that the graduate students there were the best in the country. The circumstances under which I was "called to Harvard" (an expression which, I hope, is in its sunset) were somewhat embarrassing: all of the members of the Physical Chemistry Division of the U of M were in my office. Several graduate students and staff came to Harvard with me, along with one infamous piece of apparatus: a Rigagu-Denki rotating anode X-ray unit. That very early model, purchased for about $4,000, was rebuilt by William Streib and Sandy Mathieson many times. When it finally worked I could not persuade anyone to use it because of its history. So I gave it away to the Laboratory of Applied Physics if only they would pay the cost of moving it. Shaughnessy, the movers, had to take it out of the window but they used a long beam to which they attached a rope, which broke. Just before that one of the students said, "I wonder what would happen it the rope broke?" It fell three floors, ended one foot into the solid ground, and was sold for junk. While visiting Harvard in the spring of 1959,1 talked with Roald Hoffmann about programming extended Huckel theory for the new IBM computer which I influenced Harvard to obtain. It was only after he spent a year in Moscow that he returned to start the molecular orbital work which was first successfully used on the polyhedral borane anions, and now has been applied to many parts of the periodical table, including (years later) tests during the development of the

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Woodward-Hoffmann rules. It was in 1959 that I first visited M . Frederick Hawthorne, then at Huntsville, Alabama, where we began a collaborative effort which has lasted for years. I saw the B H B nmr spectrum, and realized a few days later what the correct polyhedral structure must be, as well as its mechanism of formation from collapse of the more open (nido) framework of B H L where L is a ligand such as trimethylamine (J.A.C.S. 81, 5833 (1959)). Shortly thereafter Hawthorne gave us a salt of B H ~ which we showed had an icosahedral structure, as had been predicted by M . deV. Roberts and H. C. Longuet-Higgins, and by us. Hawthorne supplied us with many compounds in those days, and Russell Grimes made some more new ones here. It was satisfying to see the enormous development of this area of chemistry, and to feel that the theoretical and X-ray structural approaches were, for once, leading the way. I continued to look for new structural principles and to try to formulate principles of chemical reactions, partly summarized in my 1963 book "Boron Hydrides" which is long since out of print, having sold only 4000 copies. Even the hypho-boranes are foreshadowed in this book. Prepared by the earlier work on pseudorotation noted above, and by the independent interpretation by Robert E. Williams and me of pseudorotation as the mechanism for the 1 to 2 shift of substituents R in B H R, I studied this process further. I remember recalling the juxtaposition of the icosahedral B unit and the cubeoctahedral B unit in a figure in the paper published in 1960 with Doyle Britton on Valence Structure of the Higher Borides. I wondered if the cubeoctahedron could close its square faces in one or the other of two different ways, and thus give a pathway from one icosahedral to another icosahedral structure. Not long after a thought occurred to me of this same process, more locally, in B H " in which a B diamond-shaped unit could open to square, and then close the other way, to produce an isomerization. I remember Roald Hoffmann asking, "How do you think of ideas like that?" This was to become a general paper on pseudorotation mechanisms in polyhedral species (Science 153, 373 (1956)), an idea which was worked out at home with models when I was recovering from influenza. Actually the programming of extended Huckel theory was begun in my laboratory by Lawrence L. Lohr, Jr., whose interests in transition metal chemistry required d orbitals. When Hoffmann returned from Russia, he also started, but needed only s and ρ orbitals, so he finished his program first. We applied the new program to many compounds, but I received so much criticism about the basis of the theory, mostly from Ε. B. Wilson, Jr., that I suggested to Marshall Newton and F.P. Boer that they look into improving the theory. I had previously started C. William Kern, Richard M. Stevens and Russell M . Pitzer on self-consistent field theories of molecules, and of certain second order properties including the chemical shift. Although even now (1977) we have failed to calculate B chemical shifts reliably in a complex borane, we did succeed for many diatomics, finding paramagnetism in diatomic BH even 2

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though all electrons are paired. The SCF theory of the ground state was, however, applicable to large molecules. I took particular delight in asking Russell Pitzer to make the first correct calculation of the barrier to internal rotation in ethane: it was his father, Kenneth S. Pitzer, who found this barrier experimentally, before World War II. I am told that I confused theoretical chemists of that time by using relatively exact SCF theory and relatively crude extended Huckel theory. The purpose was eventually to produce a relationship which would then allow the calculation of much more reliable wave functions for large molecules and for reaction pathways. The results of the SCF theory became an objective, and we proceeded from a non-empirical theory to our latest one ( 1977), partial retention of diatomic differential overlap (PRDDO), programmed by Thomas A. Halgren. He also programmed a "synchronous transit" method for reaction pathways of internal rearrangement ring opening, or of chemical reactions between different molecules. The presence of these new theoretical methods, made possible by high speed computers, allowed us to reinvestigate the bonding in boranes, carboranes and related compounds in a totally objective way. Many of the simplest ideas of three-center bond theory were confirmed, and the limits of validity (about 0.1 to 0.2 electrons/bond) were established. Also, preferred single valence structures, sometimes possessing fractional bonds, emerged where previously we had to use many resonance structures in a hybrid description. These methods of localizing molecular orbitals (Lennard-Jones, Pople, Boys, Edmiston, Ruedenberg, all of other laboratories) greatly simplified bonding descriptions of these boron compounds. Also, they produced a vivid connection between the highly delocalized symmetry molecular orbitals and the localized bonds in which chemists believe so strongly. When I reviewed this work at the American Chemical Society on the occasion of the Debye Award, H. F. Schaefer commented to me, "Other people had looked at localized orbitals, but you are the only one to do so much chemistry with them." The work has only begun. These localized orbitals are more complex than the usual chemical bonds, and we have hardly begun to look at the delocalized "tails" which are mostly neglected, but I am convinced that these orbitals are a more accurate description of electron pair bonds than are more conventional bonds. Even so, they need drastic modification when electrons are unpaired in chemical reactions. Our studies of these localized orbitals since 1969 were mentioned by others, along with the many earlier X-ray, bonding, and chemical studies in my conversations in Sweden in December 1976. In probing for new areas, during those intervals in which boron chemistry would allow me to do so, my students and I explored several other areas related to structure and function. William E. Streib built a helium cryostat which allowed us to solve the crystal structures of β-Ν , α-Ν , P-F , γ - 0 , C H and B H . A study of (OC) FeC H gave a crystal structure in which the (OC) Fe was bonded to only four C's of the cyclooctatetraene, while all Η atoms were 2

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equivalent in the nmr spectrum. From these facts I guessed that the ring was rotating through the bonding region on the nmr time scale. This was one of the first proposals of dynamical effects in transition metal complexes (1962), but not as early as one of Geoffrey Wilkinson' s proposals. Of the organic natural products, perhaps the structure of vincristine (with J. William Moncrief, J. Am. Chem. Soc. 87, 4963 (1965)) was most important because of the previously incorrect stereochemistry, the use of anomalous scattering in the solution, and the success of this compound in the treatment of childhood leukemia. From the molecular orbitals of hypothetical B H , I once concluded that cyclobutadiene might be a tetrahedral molecule, which it is certainly not, but where else could one publish such a paper but Tetrahedron Letters? One disappointment was that the National Science Foundation refused to support the work started by J. Gerratt and me on spin-coupled wave functions (Proc. Natl. Acad. Sci. U.S. 59, 332 (1968)). Unfortunately, they also abruptly discontinued my only NSF Grant in 1964 after only four years of support of the research on boranes. Support from NSF was, however, resumed in 1977 subsequent to an event in December 1976. More recently, Daniel Jones and I studied the effect of chemical bonding on the distances in B H as determined by X-ray methods. This is a general area of direct determination of electron densities throughout a molecule, but I decided not to make a major effort in this direction because it would require complete and accurate X-ray data at the temperatures of liquid N as well neutron diffraction results on the nuclear positions. Also, for a few years, we tried to attach borane and carborane cages to organic molecules which were then to be attached to specific antibodies to tumors for treatment by neutron irradiation (/. Medicinal Chem. 17, 785 and 792 (1974)). My interest in biochemistry goes back to my perusal of medical books in my father's library and to the influence of Linus Pauling from 1942 on. After trying unsuccessfully (with Scott Matthews) to isolate a small enzyme from tetrahymena pyroformis in Minnesota days, I started the carboxypeptidase A work in 1961. However, the project did not really get under way until Martha Ludwig arrived in May of 1962. In August of 1963 we published the solution of the lead derivative, but progress slowed after that for a time. Morale problems were due to the pessimistic outlook of two members of the rather large group that assembled. In retrospect, optimism was well justified- the derivatives were good; the intensity changes were not primarily due, after all, to conformational changes. Those who stayed with the project (Martha Ludwig, Jean Hartsuck, Thomas Steitz, James Coppola, Florante Quiocho, Hilary Muirhead, George N. Reeke, Jr. and Paul Bethge) were most cooperative, and have distinguished themselves since in independent studies. Is it the nature of the cooperative research, the field of biochemistry, the dilution of individual identity, or some other factor that makes biochemists generally, but not always, more sensitive than chemists to their ambitions? I am not referring to those members of my group as listed above.


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The accomplishments on the carboxypeptidase A study were impressive. First, I located the disulfide bridge in spite of the overwhelming evidence of Vallee that there could not be one because one of the two sulfhydryl groups was bound to zinc. It was not. We identified the three ligands to zinc as 69,72 and 196 in the polypeptide chain without a sequence, and properly identified 69 as His and 72 as Glu, but misidentified His 196 as Lys. In our proposal of a mechanism, Glu 270 either attacks the substrate or promotes the attack of water, and Tyr 248 (or water) is the proton donor when peptides are cleaved. At the binding stage Arg 145 forms a doubly hydrogen bonded salt link to the Cterminal carboxylate group of the substrate. High resolution was achieved in August 1966, as reported at a Gordon Conference. Only myoglobin and lysozyme had reached high resolution at that time. It is sometimes extremely difficult to repeat a biochemical experiment, and the preparation of the crystalline form which we studied ( a= 51.41 Â, c = 47.19 Â and β = 97°35') was just such a problem. Only about two of some thirty preparations crystallized with these cell dimensions. On the other hand most preparations, including the commercial preparation, give cell dimensions of a= 50.9 Â, b = 57.9 Â, c = 45.0 A and β = 94°40\ The activities of these two forms are very different in the solid state, 1/3 and 1/300 of that of the material in solution (Proc. Nat. Acad. Sci. 70, 3797 (1973)). The arsanilzao Tyr 248 derivative of the former crystals behaves like the material in solution, but this derivative of the latter crystals is quite different from the material in solution. Vallee continues to discount the results of our crystallography, done on the former unit cell, on the basis of his results on the commercial form, which most likely has the latter unit cell. Probably the main difference is in the intermolecular contacts in the two crystalline forms. Since the crystals of the Xray study are active, the conclusion of this work should be tested on that basis. Our present plan (1977) is to elucidate the structures of the protein inhibitor of carboxypeptidase A isolated from potatoes, and of its complex with the enzyme in the hope that atomic displacements toward the transition state can be recognized. The glucagon structure was a different matter. William Haugen, not a careful experimentalist, worked only at high pH where the intensity changes upon addition of heavy atom salts were mostly conformational changes. Jean Hartsuck made that observation. The low resolution structure was noted in 1969, but it was Thomas Blundell, one of Dorothy Hodgkin's former students, who later solved the structure at a lower pH. We sent him all of our results, and I have the highest praise of him as a scientist and a gentleman. Concanavalin A, was solved here to 4 Â resolution, by Florante Quiocho. The refinements are detailed in the Ph.D. thesis of Brian Edwards, who did most of the calculations on Quiocho's data. We entered a collaboration with Gerald Edelman at Rockefeller University, where the sequence was being done and where George Reeke, Jr. was just then setting up a new laboratory for protein


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xxvi Eaton et al.; Structures and Mechanisms ACS Symposium Series; American Chemical Society: Washington, DC, 2002.

structure research. A joint publication (Proc. Nat. Acad. Sci. 68, 1853 (1971)) is based entirely on Quiocho's data. Since 1967 our major effort has gone into the determination of the various structures of an allosteric enzyme, aspartate transcarbamylase from E. coli. Our first publication has an error, due to our belief in Schachrnan's result that the enzyme was a terminer (C R ), and also due to our failure to compute the water content correctly: We found our error, and discovered both a two-fold and a three-fold axis in the molecule, and therefore the hexameric nature of this molecule (Wiley and Lipscomb, Nature 218, 1119 (1968)). Simultaneously, Klaus Weber, in his own laboratory here at Harvard, found the six-fold symmetry from analysis of the N-terminal groups, and from the sequence of the regulatory chain (R). The catalytic chain (C) has now (1977) been almost completely sequenced by William Konigsberg of Yale, who has kindly sent us his results from time to time. Thus the enzyme has the composition Q R ^ where C is about 34,000 and R is about 17,000 in molecular weight. The work is at about 3 Â resolution for the native enzyme and for its complex with CTP (Cytidine triphosphate is the allosteric inhibitor); and crystals have been obtained of the complex of this enzyme with a substrate analogue. Finally, this problem which may go on for more years, is moving well.


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Why was it so difficult earlier? The major problem was to obtain at least one derivative in which the heavy atom could be located. The problems were (1) the high molecular weight (310,000) and hexameric nature which made it difficult to locate heavy atoms in multiple site derivatives, (2) the sensitivity of the enzyme to conformational changes which destroyed the single crystal, and (3) the greater reactivity (destroying the crystal) of the sulfhydryls of the regulatory chain as compared with that of the single sulfhydryl of each catalytic chain. It was Cecil McMurray who solved this problem by making a large number of closely related mercurials, of which 2-chloromercuri-4-nitrophenol was most successful in its preference for the sulfhydryl of the catalytic chain. With one heavy atom per C chain, we were then able to solve for the heavy atom positions in the uranyl, platinum and gold derivatives. Even now there are problems because the X-ray data fade out at about 3 Â and because our experimental intensities have a background which is too high. Also, of these four derivatives, only the Hg and U are good to 3 Â resolution, so that we shall have to use averaging of non-crystallographic symmetry in order to improve the phasing of the diffraction maxima. However, the research problem is well worth the effort. The enzyme shows sigmoidal kinetics indicating positive cooperativity, inhibition by cytidine triphosphate, stimulation by adenosine triphosphate, and negative cooperativity in binding of CTP. Also, Elita Pastra-Landis and David Evans have shown here that the fragment Q R 4 has reduced, but very definite cooperativity. The distance between the allosteric (regulatory) site and the catalytic site is 43 Â, an amazingly long distance for propagation of an allosteric signal. Also, the

xxvii Eaton et al.; Structures and Mechanisms ACS Symposium Series; American Chemical Society: Washington, DC, 2002.

mechanisms of catalysis of the reaction of carbamyl phosphate with aspartate to give carbamyl aspartate, the first step of the pyrimidine pathway, is still a mystery. Although the theme of X-ray crystallographic studies of structures runs through much of this account, it has been of great interest to us to carry on parallel experimental, computer or theoretical studies in order to illuminate the relationships of the structures to the rest of the chemistry. On occasion this diversity has even helped the research. The excellent crystals of unliganded aspartate transcarbamylase were obtained by accident from an nmr study during a kinetics experiment. Certainly, the interests of the research group have been broadened, and I have normally been able to ask for a Ph.D. thesis in more than one technique on a particular subject. In a real sense, we have thus generated our own research problems, rather than starting from the open literature. Seemingly unrelated areas come together, for example our recent attempts to use molecular orbital theory to elucidate the sequential steps in a model of the active site of an enzyme.


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Acknowledgments I wish to thank the author for permission to add three footnotes to the original 1977 manuscript.

Footnotes 1

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However, see B. Post, Phys. Rev. Lett. 39, 760 (1977). In 1982 D. C. Rees and I added Glu 270 as a possible proton donor (J. Mol. Biol. 160, 475). See J. E. Gouaux, K. L. Krause and W. N. Lipscomb, Biochem. Biophys. Research Commun. 142, 893 (1987).

xxviii Eaton et al.; Structures and Mechanisms ACS Symposium Series; American Chemical Society: Washington, DC, 2002.

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